The long-term objectives of this research proposal is to elucidate the signal transduction cascade associated with Fas mediated apoptosis in mammalian cells. Several disease states have been associated with the disruption of this pathway, including several types of cancers that express a negative regulatory protein (FAP-1) for Fas-mediated apoptosis, Neimann-Pick Disease (loss of acidic sphingomyelinase activity), and recently identified syndromes of autoimmunity related to point mutation in the Fas gene. Using cells from Neimann-Pick patients, "Knockout mice" deficient in acidic sphingomyelinase, and expression vectors with human acidic sphingomyelinase, FAP-1, and FADD, we will attempt to discover the sequential mechanism of FAS-mediated apoptosis from the engagement of the Fas molecule to the production of ceramide from sphingomyelin. We will use cell cytotoxicity assays with anti-Fas antibody and ceramide, assays of sphingomyelinase activity and upregulation, and measurements of RAS activation to follow the progress (or inhibition) of the signal for cell death. A greater understanding of the signal transduction cascade and naturally occurring diseases caused by disruptions in this pathway will pave the way for possible therapies based on gene transfer or improved chemotherapy strategies.